miRNA介导选择性表达肿瘤坏死因子相关的凋亡诱导配体抑制肾癌细胞生长
摘要
肾细胞癌是常见的肾脏肿瘤，占肾脏肿瘤的第三位，其发病率逐年增高，且有易发于老年，男性较女性更为常见。肾细胞癌治疗主要依赖手术及腹腔镜等手段，故大多数患者常被术后并发症以及放化疗的副作用所困扰，大大降低了生活质量。因此寻找特异的治疗靶点及治疗手段十分重要。肿瘤坏死因子相关凋亡诱导配体（tumor necrosisfactor-related apoptosis-inducing ligand，TRAIL）做为TNF家族成员，能够诱导肾细胞癌肿瘤细胞凋亡。本研究的目的在于通过miRNA反应元件MRE，调控TRAIL在癌症细胞组织与正常细胞组织中的表达，从而选择性杀伤肿瘤细胞，对正常组织器官不产生细胞毒性。具体研究如下：
1实验路线
1.1miR-138, miR-199在肾癌细胞系、组织中与正常细胞系、组织中的表达。
1.1.1HK-2人正常肾上皮细胞系，ACHN和786-O细胞系和L-02人正常肝细胞培养。
1.1.2通过实时定量PCR比较RCC细胞系和正常细胞系miR-138和miR-199表达水平的差异。
1.1.3收集患者的肾癌和正常肾组织新鲜组织。
1.1.4通过实时定量PCR检测比较肾细胞癌组织和正常肾组织中miR-138，miR-199表达水平的差异。
1.2在miR-138，miR-199的MRE的控制下，RCC细胞外源基因的表达。
1.2.1通过将这些的MRE的两个拷贝到荧光素酶的载体编码区的下游部位，产生psiCheck2-3MREs。
1.2.2psiCheck2和psiCheck2-3MREs的转染HK-2，L-02，ACHN和786-O细胞，48小时后确定用双荧光素酶报告系统确定其荧光素酶活性。
1.3TRAIL在RCC细胞内的腺病毒载体下miR-138，miR-199的MRE的调节介导的表达。
1.3.1将miR-138，miR-199的MRE的两个拷贝放入开放阅读框后面的生成AD-TRAIL-3MREs。
1.3.2免疫印迹比较Ad-TRAIL，AD-TRAIL-3MREs与Ad-EGFP转染后，在L-02和ACHN上TRAIL表达的差异。
1.3.3实时定量PCR比较Ad-TRAIL，AD-TRAIL-3MREs与Ad-EGFP转染后，在L-02肝细胞和ACHN上TRAIL表达的差异。
1.4miR-138, miR-199在细胞内调控腺病毒载体，进而影响TRAIL在RCC细胞中的表达。
1.4.1通过免疫印迹和实时定量PCR比较转染miR-138，miR-199和抑制剂L-02之间TRAIL表达水平差异。
1.4.2通过免疫印迹和实时定量PCR比较转染miR-138，miR-199和抑制剂ACHN之间TRAIL表达水平差异。
1.5western blot和G0/G1细胞比值检测比较由Ad-TRAIL-3MREs诱导ACHN细胞和L-02细胞的凋亡。
1.6MTT检测在各种重组腺病毒感染下，各种RCC细胞和正常细胞的活性。
1.7在各种重组腺病毒感染下，检测动物体内ACHN诱发的RCC肿瘤生长情况，在RCC肿瘤组织中TRAIL和凋亡转导通路的表达
1.8调查TRAIL诱导的肝细胞毒性。
1.8.1从无肿瘤但给予PBS和重组腺病毒小鼠身上获取血液和血清。
1.8.2ELISA检测血ALT水平
1.8.3western blot检测给予重组腺病毒后肝组织TRAIL和凋亡转导通路的表达。
2结果
2.1评价RCC和正常细胞miR-138，miR-199的表达水平。与正常对照组比较，RCC组织和RCC细胞系中，miR-138，miR-199被认为是低表达。在正常肾细胞HEK-293和肝细胞L-02中，miR-138，miR-199过表达。
2.2miR-138，miR-199的MRE限制正常细胞内外源基因的表达。
与psiCheck2转染组相比，转染psiCheck2-3MREs组的HEK-293和L-02细胞的荧光素酶表达水平被大大地抑制。然而，转染psiCheck2和psiCheck2-3MREs RCC细胞系之间的荧光素酶活性没有显著差异。
2.3L-02和ACHN细胞转染Ad-TRAIL, Ad-TRAIL-3MREs和Ad-EGFP后，检测TRAIL蛋白的表达。
AD-TRAIL转染的L-02和ACHN细胞表达的TRAIL能力没有差别。与此相反，在Ad-TRAIL-3MREs感染的L-02细胞，TRAIL的表达被大大地抑制，但在ACHN细胞TRAIL表达则无影响。 qPCR实验进一步证实，TRAIL的mRNA可以在Ad-TRAIL和Ad-TRAIL-3MREs感染的ACHN细胞以及Ad-TRAIL感染L-02细胞很容易地检测到。但在转染Ad-TRAIL-3MREs正常肝细胞无TRAIL mRNA表达。
2.4由于miR-138，miR-199在细胞内调节， Ad-TRAIL-3MREs转染的RCC细胞系中选择性表达TRAIL。
免疫印迹和qPCR实验表明，转染miR-138，miR-199抑制剂L-02细胞部分恢复TRAIL的表达。由于转染了miRNA相似物，增加了miR-138，miR-199的水平，ACHN肾癌细胞表达TRAIL适度降低。
2.5MRE调控腺病毒载体选择性表达TRAIL导致肾癌细胞的选择性凋亡。
在Ad-TRAIL感染L-02细胞和Ad-TRAIL或Ad-TRAIL-3MREs转染ACHN细胞上裂解的Caspase3和PARP蛋白的高表达。Ad-TRAIL-3MREs转染的L-02转导的细胞Caspase3和PARP没有改变。
Ad-TRAIL感染L-02细胞和用Ad-TRAIL或Ad-TRAIL-3MREs转染的ACHN细胞中sub-G0/G1细胞的比值增加。然而，Ad-TRAIL-3MREs转染的L-02细胞sub-G0/G1的比例是相当低的。
2.6Ad-TRAIL-3MREs能够诱导RCC细胞的细胞毒性而不损伤正常细胞。
这些数据表明，AD-TRAIL在降低癌细胞和正常细胞的生存没有任何歧视。与此相反，AD-TRAIL-3MREs在诱导RCC细胞毒性时有一个高选择性。
2.7在体内生长的RCC可以通过AD-TRAIL-3MREs治疗来抑制。
用Ad-TRAIL和Ad-TRAIL-3MREs治疗的小鼠中，极大地抑制ACHN异种移植的肿瘤。TRAIL表达的免疫印迹分析表明，注射Ad-TRAIL和Ad-TRAIL-3MREs肿瘤之间TRAIL的表达无显著差异。另外，在两组肿瘤中检测出caspase3和PARP的裂解。
2.8Ad-TRAIL-3RMEs能够保护肝脏避免TRAIL诱导的凋亡。
用Ad-TRAIL治疗的小鼠ALT水平显著提高。与用Ad-EGFP和PBS对照相比，用Ad-TRAIL-3MREs治疗的小鼠血清ALT水平没有发生变化。此外，TRAIL与细胞凋亡相关蛋白的免疫印迹分析表明，AD-TRAIL激活肝组织凋亡途径，而AD-TRAIL-3MREs没有影响肝细胞的状态。
3结论
3.1在RCC细胞和组织与正常的人相比较miR-138，miR-199的水平降低。
3.2miR-138，miR-199的MRE可以有效地抑制在正常细胞外源基因的表达。
3.3miR-138，miR-199的MRE可以用于限制在RCC细胞腺病毒介导的TRAIL的表达。
3.4miR-138，miR-199的水平控制腺病毒载体介导的TRAIL选择性表达。
3.5TRAIL选择性表达能够诱导RCC特异性凋亡。
3.6MRE调节的腺病毒是对正常细胞无细胞毒性的，有针对性的抗肿瘤剂。
3.7Ad-TRAIL-3MREs能够经由TRAIL诱导的细胞凋亡抑制的RCC肿瘤在小鼠中的生长。
3.8MRE调控TRAIL表达避免了TRAIL诱导的肝毒性。
Renal cell carcinoma accounts for third place of kidney cancer. Theincidence of RCC increased year by year, and there is prone in older,more common in men than women. Treatment is mainly dependent onrenal cell carcinoma and laparoscopic surgery etc, which often causedpostoperative complications and side effects of chemotherapy, greatlyreduce the quality of the life of patient. Therefore, finding noveltherapeutic targets and specific treatment is very important. Tumornecrosis factor-related apoptosis-inducing ligand (TRAIL) as a memberof the TNF family is capable of inducing apoptosis in renal cellcarcinoma. The purpose of this study is under the control of miRNAresponse element MRE, regulates the expression of TRAIL in cancertissue and normal tissue cells, thereby selectively killing tumor cells,without damage of normal tissues and organs. Specific studies are asfollows:
1. Experimental Routes:
1.1Expression of miR-138, miR-199within RCC cell line and tissuevs. normal cell line and tissue.
1.1.1HK-2human normal kidney epithelium cell line, ACHN and786-O RCC cell lines and L-02human normal liver cells were cultured
1.1.2Compare the differences of miR-138, miR-199expression level ofRCC cell line and normal via Real-time quantitative PCR.
1.1.3Collected fresh tissue of patients with RCC and normal renaltissues.
1.1.4Compare the differences of miR-138, miR-199and expressionlevel of RCC tissue and normal renal tissue via Real-time quantitativePCR.
1.2Expression of exogenous genes within RCC cells under thecontrol of MREs of miR-138, miR-199.
1.2.1psiCheck2was reconstructed by inserting two copies of theseMREs into the downstream sites of luciferase coding region on thevectors, generating psiCheck2-3MREs.
1.2.2Luciferase activity was determined in HK-2, L-02, ACHN and786-O cells,48h after the transfection of psiCheck2andpsiCheck2-3MREs, with Dual-Luciferase reporter system
1.3Expression of TRAIL mediated by adenoviral vectors withinRCC cells under the regulation of MREs of miR-138, miR-199.
1.3.1Ad-TRAIL-3MREs were generated with two copies of MREs ofmiR-138, miR-199immediately following the open reading frame.
1.3.2Compare difference of TRAIL expression in L-02, and ACHNtransfected with Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFP viawestern blot.
1.3.3Compare difference of TRAIL expression in L-02, ACHN andliver cell transfected with Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFPvia Real-time quantitative PCR.
1.4Expression of TRAIL mediated by adenoviral vectors withinRCC cells under the regulation of abundance of miR-138, miR-199and miR-122.
1.4.1Compare the differences TRAIL levels between L-02transfectedwith the inhibitor of miR-138, miR-199via western blot and Real-timequantitative PCR.
1.4.2Compare the differences TRAIL levels between ACHN transfected with the mimics of miR-138, miR-199western blot and Real-timequantitative PCR.
1.5Evaluate the apoptosis pathway (western bolt) and portion ofG0/G1cells of ACHN cells and L-02cells induced by Ad-TRAIL-3MREs.
1.6MTT assay to examine the viability of RCC cell lines and normalcell lines, under the infection of recombinant adenoviruses.
1.7Exam the growth of RCC tumors in mice by TRAIL-inducedapoptosis via the growth of ACTH, the expression of TRAIL and theapoptosis pathway under the injection of recombinant adenoviruses.
1.8Investigate the hepatic tissue from TRAIL-induced cytotoxicity.
1.8.1Harvest the blood and serum from the mice bear no tumoradministrated by PBS and recombinant adenoviruses.
1.8.2Blood ALT level was evaluated by ELISA.
1.8.3Exam the expression of TRAIL and the apoptotic pathway in livertissue administrated by recombinant adenoviruses via western blot.
2. Result
2.1evaluate the expression levels of miR-138, miR-199betweenRCC and normal cells.
In primary RCC samples and RCC cell lines, miR-138, miR-199was found to be underexpressed, in comparison with normal control.miR-138, miR-199were overexpressed in normal kidney cells,HEK-293, and hepatic cells, L-02.
2.2MREs of miR-138, miR-199and restrict the expression ofexogenous genes within RCC cells.
The expression level of luciferase was greatly suppressed inHEK-293, and L-02transfected with psiCheck2-3MREs, compared with psiCheck2-transfected ones. However, there was no significantdifference in luciferase activity between RCC cell lines transfected withpsiCheck2and psiCheck2-3MREs.
2.3Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFP were added to theculture of L-02and ACHN cells, followed by the detection of TRAILprotein expression.
The data indicated that Ad-TRAIL has no difference in its ability toexpress TRAIL between L-02and ACHN cells. In contrast, TRAILexpression was greatly suppressed in Ad-TRAIL-3MREs-infected L-02cells, but not in ACHN cells. qPCR assays further confirmed thatTRAIL mRNAs can be easily detected in Ad-TRAIL-andAd-TRAIL-3MREs-infected ACHN cells as well as Ad-TRAIL-infectedL-02cells. But there was no TRAIL mRNA in normal liver cellstransduced with Ad-TRAIL-3MREs.
2.4The selective expression of TRAIL in Ad-TRAIL-3MREs-infected RCC cell lines is due to the regulation of miR-138, miR-199in cells.
The immunoblot and qPCR assays indicated that TRAILexpression was partially restored in L-02cells transfected with theinhibitors of miR-138, miR-199. Accordingly, TRAIL expression wasmoderately reduced in ACHN RCC cells, which has increased levels ofmiR-138, miR-199due to the transfection of miRNA mimics.
2.5The selective expression of TRAIL mediated by MREs-regulatedadenoviral vector can also lead to selective apoptosis in RCC cells.
The results revealed that cleaved caspase3and PARP proteinswere highly expressed in Ad-TRAIL-infected L-02cells and ACHNcells treated with Ad-TRAIL or Ad-TRAIL-3MREs. In contrast, therewas no cleavage of caspase3and PARP in L-02cells transduced with Ad-TRAIL-3MREs.
Increased portion of sub-G0/G1cells was observed in inAd-TRAIL-infected L-02cells and ACHN cells treated with Ad-TRAILor Ad-TRAIL-3MREs. However, the percentage of sub-G0/G1is quitelow in the L-02cell infected with Ad-TRAIL-3MREs.
2.6Ad-TRAIL-3MREs was able to induce cytotoxicity to RCC cellswithout damage normal cells.
The data indicated that Ad-TRAIL has no discrimination inreducing the survival of cancer cells and normal cells. In contrast,Ad-TRAIL-3MREs exerts a high selectivity to RCC cells in inducingcytotoxicity.
2.7The in vivo growth of RCC can be inhibited by Ad TRAIL-3MREs treatment
The growth of ACHN xenograft was greatly suppressed in the micetreated with Ad-TRAIL and Ad-TRAIL-3MREs. Furthermore,immunoblot analysis of TRAIL expression revealed that there was nosignificant difference in TRAIL expression between the tumors injectedwith Ad-TRAIL and Ad-TRAIL-3MREs. Also, the cleavage of caspase3and PARP was detected in the two groups of tumor.
2.8Ad-TRAIL-3RMEs can protect liver from TRAIL-inducedapoptosis
ALT level was significantly heightened in the mice treated withAd-TRAIL. In contrast, serum ALT levels did not change in the micetreated with Ad-TRAIL-3MREs, in comparison with Ad-EGFP and PBS.Furthermore, immunoblot analysis of TRAIL and apoptosis-relatedproteins indicated that Ad-TRAIL activated the apoptotic pathway inliver tissue, whereas Ad-TRAIL-3MREs did not affect the apoptotic
3. Conclusions
3.1The level of miR-138, miR-199was reduced in RCC cells and tissuecompared with the normal ones.
3.2MREs of miR-138, miR-199can effectively suppress the expressionof exogenous genes in normal cells.
3.3MREs of miR-138, miR-199can be used for restrictadenovirus-mediated TRAIL expression in RCC cells.
3.4The selective expression of TRAIL mediated by adenoviral vectorresulted from the levels of miR-138, miR-199.
3.5The selective TRAIL expressions were able to induce RCC-specificapoptotic event.
3.6MREs-regulated adenovirus is a targeted anti-tumor agent withoutcytotoxicity to normal cells.
3.7Ad-TRAIL-3MREs was able to suppress the growth of RCC tumorsin mice via TRAIL-induced apoptosis.
3.8MREs-regulated TRAIL expression protected liver from TRAIL-induced toxicity.
1实验路线
1.1miR-138, miR-199在肾癌细胞系、组织中与正常细胞系、组织中的表达。
1.1.1HK-2人正常肾上皮细胞系，ACHN和786-O细胞系和L-02人正常肝细胞培养。
1.1.2通过实时定量PCR比较RCC细胞系和正常细胞系miR-138和miR-199表达水平的差异。
1.1.3收集患者的肾癌和正常肾组织新鲜组织。
1.1.4通过实时定量PCR检测比较肾细胞癌组织和正常肾组织中miR-138，miR-199表达水平的差异。
1.2在miR-138，miR-199的MRE的控制下，RCC细胞外源基因的表达。
1.2.1通过将这些的MRE的两个拷贝到荧光素酶的载体编码区的下游部位，产生psiCheck2-3MREs。
1.2.2psiCheck2和psiCheck2-3MREs的转染HK-2，L-02，ACHN和786-O细胞，48小时后确定用双荧光素酶报告系统确定其荧光素酶活性。
1.3TRAIL在RCC细胞内的腺病毒载体下miR-138，miR-199的MRE的调节介导的表达。
1.3.1将miR-138，miR-199的MRE的两个拷贝放入开放阅读框后面的生成AD-TRAIL-3MREs。
1.3.2免疫印迹比较Ad-TRAIL，AD-TRAIL-3MREs与Ad-EGFP转染后，在L-02和ACHN上TRAIL表达的差异。
1.3.3实时定量PCR比较Ad-TRAIL，AD-TRAIL-3MREs与Ad-EGFP转染后，在L-02肝细胞和ACHN上TRAIL表达的差异。
1.4miR-138, miR-199在细胞内调控腺病毒载体，进而影响TRAIL在RCC细胞中的表达。
1.4.1通过免疫印迹和实时定量PCR比较转染miR-138，miR-199和抑制剂L-02之间TRAIL表达水平差异。
1.4.2通过免疫印迹和实时定量PCR比较转染miR-138，miR-199和抑制剂ACHN之间TRAIL表达水平差异。
1.5western blot和G0/G1细胞比值检测比较由Ad-TRAIL-3MREs诱导ACHN细胞和L-02细胞的凋亡。
1.6MTT检测在各种重组腺病毒感染下，各种RCC细胞和正常细胞的活性。
1.7在各种重组腺病毒感染下，检测动物体内ACHN诱发的RCC肿瘤生长情况，在RCC肿瘤组织中TRAIL和凋亡转导通路的表达
1.8调查TRAIL诱导的肝细胞毒性。
1.8.1从无肿瘤但给予PBS和重组腺病毒小鼠身上获取血液和血清。
1.8.2ELISA检测血ALT水平
1.8.3western blot检测给予重组腺病毒后肝组织TRAIL和凋亡转导通路的表达。
2结果
2.1评价RCC和正常细胞miR-138，miR-199的表达水平。与正常对照组比较，RCC组织和RCC细胞系中，miR-138，miR-199被认为是低表达。在正常肾细胞HEK-293和肝细胞L-02中，miR-138，miR-199过表达。
2.2miR-138，miR-199的MRE限制正常细胞内外源基因的表达。
与psiCheck2转染组相比，转染psiCheck2-3MREs组的HEK-293和L-02细胞的荧光素酶表达水平被大大地抑制。然而，转染psiCheck2和psiCheck2-3MREs RCC细胞系之间的荧光素酶活性没有显著差异。
2.3L-02和ACHN细胞转染Ad-TRAIL, Ad-TRAIL-3MREs和Ad-EGFP后，检测TRAIL蛋白的表达。
AD-TRAIL转染的L-02和ACHN细胞表达的TRAIL能力没有差别。与此相反，在Ad-TRAIL-3MREs感染的L-02细胞，TRAIL的表达被大大地抑制，但在ACHN细胞TRAIL表达则无影响。 qPCR实验进一步证实，TRAIL的mRNA可以在Ad-TRAIL和Ad-TRAIL-3MREs感染的ACHN细胞以及Ad-TRAIL感染L-02细胞很容易地检测到。但在转染Ad-TRAIL-3MREs正常肝细胞无TRAIL mRNA表达。
2.4由于miR-138，miR-199在细胞内调节， Ad-TRAIL-3MREs转染的RCC细胞系中选择性表达TRAIL。
免疫印迹和qPCR实验表明，转染miR-138，miR-199抑制剂L-02细胞部分恢复TRAIL的表达。由于转染了miRNA相似物，增加了miR-138，miR-199的水平，ACHN肾癌细胞表达TRAIL适度降低。
2.5MRE调控腺病毒载体选择性表达TRAIL导致肾癌细胞的选择性凋亡。
在Ad-TRAIL感染L-02细胞和Ad-TRAIL或Ad-TRAIL-3MREs转染ACHN细胞上裂解的Caspase3和PARP蛋白的高表达。Ad-TRAIL-3MREs转染的L-02转导的细胞Caspase3和PARP没有改变。
Ad-TRAIL感染L-02细胞和用Ad-TRAIL或Ad-TRAIL-3MREs转染的ACHN细胞中sub-G0/G1细胞的比值增加。然而，Ad-TRAIL-3MREs转染的L-02细胞sub-G0/G1的比例是相当低的。
2.6Ad-TRAIL-3MREs能够诱导RCC细胞的细胞毒性而不损伤正常细胞。
这些数据表明，AD-TRAIL在降低癌细胞和正常细胞的生存没有任何歧视。与此相反，AD-TRAIL-3MREs在诱导RCC细胞毒性时有一个高选择性。
2.7在体内生长的RCC可以通过AD-TRAIL-3MREs治疗来抑制。
用Ad-TRAIL和Ad-TRAIL-3MREs治疗的小鼠中，极大地抑制ACHN异种移植的肿瘤。TRAIL表达的免疫印迹分析表明，注射Ad-TRAIL和Ad-TRAIL-3MREs肿瘤之间TRAIL的表达无显著差异。另外，在两组肿瘤中检测出caspase3和PARP的裂解。
2.8Ad-TRAIL-3RMEs能够保护肝脏避免TRAIL诱导的凋亡。
用Ad-TRAIL治疗的小鼠ALT水平显著提高。与用Ad-EGFP和PBS对照相比，用Ad-TRAIL-3MREs治疗的小鼠血清ALT水平没有发生变化。此外，TRAIL与细胞凋亡相关蛋白的免疫印迹分析表明，AD-TRAIL激活肝组织凋亡途径，而AD-TRAIL-3MREs没有影响肝细胞的状态。
3结论
3.1在RCC细胞和组织与正常的人相比较miR-138，miR-199的水平降低。
3.2miR-138，miR-199的MRE可以有效地抑制在正常细胞外源基因的表达。
3.3miR-138，miR-199的MRE可以用于限制在RCC细胞腺病毒介导的TRAIL的表达。
3.4miR-138，miR-199的水平控制腺病毒载体介导的TRAIL选择性表达。
3.5TRAIL选择性表达能够诱导RCC特异性凋亡。
3.6MRE调节的腺病毒是对正常细胞无细胞毒性的，有针对性的抗肿瘤剂。
3.7Ad-TRAIL-3MREs能够经由TRAIL诱导的细胞凋亡抑制的RCC肿瘤在小鼠中的生长。
3.8MRE调控TRAIL表达避免了TRAIL诱导的肝毒性。
Renal cell carcinoma accounts for third place of kidney cancer. Theincidence of RCC increased year by year, and there is prone in older,more common in men than women. Treatment is mainly dependent onrenal cell carcinoma and laparoscopic surgery etc, which often causedpostoperative complications and side effects of chemotherapy, greatlyreduce the quality of the life of patient. Therefore, finding noveltherapeutic targets and specific treatment is very important. Tumornecrosis factor-related apoptosis-inducing ligand (TRAIL) as a memberof the TNF family is capable of inducing apoptosis in renal cellcarcinoma. The purpose of this study is under the control of miRNAresponse element MRE, regulates the expression of TRAIL in cancertissue and normal tissue cells, thereby selectively killing tumor cells,without damage of normal tissues and organs. Specific studies are asfollows:
1. Experimental Routes:
1.1Expression of miR-138, miR-199within RCC cell line and tissuevs. normal cell line and tissue.
1.1.1HK-2human normal kidney epithelium cell line, ACHN and786-O RCC cell lines and L-02human normal liver cells were cultured
1.1.2Compare the differences of miR-138, miR-199expression level ofRCC cell line and normal via Real-time quantitative PCR.
1.1.3Collected fresh tissue of patients with RCC and normal renaltissues.
1.1.4Compare the differences of miR-138, miR-199and expressionlevel of RCC tissue and normal renal tissue via Real-time quantitativePCR.
1.2Expression of exogenous genes within RCC cells under thecontrol of MREs of miR-138, miR-199.
1.2.1psiCheck2was reconstructed by inserting two copies of theseMREs into the downstream sites of luciferase coding region on thevectors, generating psiCheck2-3MREs.
1.2.2Luciferase activity was determined in HK-2, L-02, ACHN and786-O cells,48h after the transfection of psiCheck2andpsiCheck2-3MREs, with Dual-Luciferase reporter system
1.3Expression of TRAIL mediated by adenoviral vectors withinRCC cells under the regulation of MREs of miR-138, miR-199.
1.3.1Ad-TRAIL-3MREs were generated with two copies of MREs ofmiR-138, miR-199immediately following the open reading frame.
1.3.2Compare difference of TRAIL expression in L-02, and ACHNtransfected with Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFP viawestern blot.
1.3.3Compare difference of TRAIL expression in L-02, ACHN andliver cell transfected with Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFPvia Real-time quantitative PCR.
1.4Expression of TRAIL mediated by adenoviral vectors withinRCC cells under the regulation of abundance of miR-138, miR-199and miR-122.
1.4.1Compare the differences TRAIL levels between L-02transfectedwith the inhibitor of miR-138, miR-199via western blot and Real-timequantitative PCR.
1.4.2Compare the differences TRAIL levels between ACHN transfected with the mimics of miR-138, miR-199western blot and Real-timequantitative PCR.
1.5Evaluate the apoptosis pathway (western bolt) and portion ofG0/G1cells of ACHN cells and L-02cells induced by Ad-TRAIL-3MREs.
1.6MTT assay to examine the viability of RCC cell lines and normalcell lines, under the infection of recombinant adenoviruses.
1.7Exam the growth of RCC tumors in mice by TRAIL-inducedapoptosis via the growth of ACTH, the expression of TRAIL and theapoptosis pathway under the injection of recombinant adenoviruses.
1.8Investigate the hepatic tissue from TRAIL-induced cytotoxicity.
1.8.1Harvest the blood and serum from the mice bear no tumoradministrated by PBS and recombinant adenoviruses.
1.8.2Blood ALT level was evaluated by ELISA.
1.8.3Exam the expression of TRAIL and the apoptotic pathway in livertissue administrated by recombinant adenoviruses via western blot.
2. Result
2.1evaluate the expression levels of miR-138, miR-199betweenRCC and normal cells.
In primary RCC samples and RCC cell lines, miR-138, miR-199was found to be underexpressed, in comparison with normal control.miR-138, miR-199were overexpressed in normal kidney cells,HEK-293, and hepatic cells, L-02.
2.2MREs of miR-138, miR-199and restrict the expression ofexogenous genes within RCC cells.
The expression level of luciferase was greatly suppressed inHEK-293, and L-02transfected with psiCheck2-3MREs, compared with psiCheck2-transfected ones. However, there was no significantdifference in luciferase activity between RCC cell lines transfected withpsiCheck2and psiCheck2-3MREs.
2.3Ad-TRAIL, Ad-TRAIL-3MREs and Ad-EGFP were added to theculture of L-02and ACHN cells, followed by the detection of TRAILprotein expression.
The data indicated that Ad-TRAIL has no difference in its ability toexpress TRAIL between L-02and ACHN cells. In contrast, TRAILexpression was greatly suppressed in Ad-TRAIL-3MREs-infected L-02cells, but not in ACHN cells. qPCR assays further confirmed thatTRAIL mRNAs can be easily detected in Ad-TRAIL-andAd-TRAIL-3MREs-infected ACHN cells as well as Ad-TRAIL-infectedL-02cells. But there was no TRAIL mRNA in normal liver cellstransduced with Ad-TRAIL-3MREs.
2.4The selective expression of TRAIL in Ad-TRAIL-3MREs-infected RCC cell lines is due to the regulation of miR-138, miR-199in cells.
The immunoblot and qPCR assays indicated that TRAILexpression was partially restored in L-02cells transfected with theinhibitors of miR-138, miR-199. Accordingly, TRAIL expression wasmoderately reduced in ACHN RCC cells, which has increased levels ofmiR-138, miR-199due to the transfection of miRNA mimics.
2.5The selective expression of TRAIL mediated by MREs-regulatedadenoviral vector can also lead to selective apoptosis in RCC cells.
The results revealed that cleaved caspase3and PARP proteinswere highly expressed in Ad-TRAIL-infected L-02cells and ACHNcells treated with Ad-TRAIL or Ad-TRAIL-3MREs. In contrast, therewas no cleavage of caspase3and PARP in L-02cells transduced with Ad-TRAIL-3MREs.
Increased portion of sub-G0/G1cells was observed in inAd-TRAIL-infected L-02cells and ACHN cells treated with Ad-TRAILor Ad-TRAIL-3MREs. However, the percentage of sub-G0/G1is quitelow in the L-02cell infected with Ad-TRAIL-3MREs.
2.6Ad-TRAIL-3MREs was able to induce cytotoxicity to RCC cellswithout damage normal cells.
The data indicated that Ad-TRAIL has no discrimination inreducing the survival of cancer cells and normal cells. In contrast,Ad-TRAIL-3MREs exerts a high selectivity to RCC cells in inducingcytotoxicity.
2.7The in vivo growth of RCC can be inhibited by Ad TRAIL-3MREs treatment
The growth of ACHN xenograft was greatly suppressed in the micetreated with Ad-TRAIL and Ad-TRAIL-3MREs. Furthermore,immunoblot analysis of TRAIL expression revealed that there was nosignificant difference in TRAIL expression between the tumors injectedwith Ad-TRAIL and Ad-TRAIL-3MREs. Also, the cleavage of caspase3and PARP was detected in the two groups of tumor.
2.8Ad-TRAIL-3RMEs can protect liver from TRAIL-inducedapoptosis
ALT level was significantly heightened in the mice treated withAd-TRAIL. In contrast, serum ALT levels did not change in the micetreated with Ad-TRAIL-3MREs, in comparison with Ad-EGFP and PBS.Furthermore, immunoblot analysis of TRAIL and apoptosis-relatedproteins indicated that Ad-TRAIL activated the apoptotic pathway inliver tissue, whereas Ad-TRAIL-3MREs did not affect the apoptotic
3. Conclusions
3.1The level of miR-138, miR-199was reduced in RCC cells and tissuecompared with the normal ones.
3.2MREs of miR-138, miR-199can effectively suppress the expressionof exogenous genes in normal cells.
3.3MREs of miR-138, miR-199can be used for restrictadenovirus-mediated TRAIL expression in RCC cells.
3.4The selective expression of TRAIL mediated by adenoviral vectorresulted from the levels of miR-138, miR-199.
3.5The selective TRAIL expressions were able to induce RCC-specificapoptotic event.
3.6MREs-regulated adenovirus is a targeted anti-tumor agent withoutcytotoxicity to normal cells.
3.7Ad-TRAIL-3MREs was able to suppress the growth of RCC tumorsin mice via TRAIL-induced apoptosis.
3.8MREs-regulated TRAIL expression protected liver from TRAIL-induced toxicity.
引文
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